Differential Ca2+ signaling characteristics of inhibitory and excitatory myenteric motor neurons in culture

Physiological studies on functionally identified myenteric neurons are scar ce because of technical limitations. We combined retrograde labeling, cell culturing, and fluorescent intracellular Ca2+ concentration ([Ca2+](i)) sig naling to study excitatory neurotransmitter responsiveness of myenteric mot or neurons. 1,1-Didodecyl-3,3,3',3'-tetramethyl indocarbocyanine (DiI) was used to label circular muscle motor neurons of the guinea pig ileum. DiI-la beled neurons were easily detectable in cultures prepared from these segmen ts. The excitatory neurotransmitters (10(-5) M) acetylcholine, substance P, and serotonin induced a transient rise in [Ca2+](i) in subsets of DiI-labe led neurons (66.7, 56.5, and 84.3%, respectively). DiI-labeled motor neuron s were either inhibitory (23.8%) or excitatory (76.2%) as assessed by stain ing for nitric oxide synthase or choline acetyltransferase. Compared with e xcitatory motor neurons, significantly fewer inhibitory neurons in culture responded to acetylcholine (0 vs. 69%) and substance P (12.5 vs. 69.2%). We conclude that combining retrograde labeling and Ca2+ imaging allows identi fication of differential receptor expression in functionally identified neu rons in culture.